Compound and method for treating myotonic dystrophy

ABSTRACT

An antisense compound for use in treating myotonic dystrophy DM1 or DM2, a method of enhancing antisense targeting to heart and quadricep muscles, and a method for treating DM1 or DM2 in a mammalian subject are disclosed. The oligonucleotide has 8-30 bases, with at least 8 contiguous bases being complementary to the polyCUG or polyCCUG repeats in the 3′UTR region of dystrophia myotonica protein kinase (DMPK) mRNA in DM1 or DM2, respectively. Conjugated to the oligonucleotide is a cell-penetrating peptide having the sequence (RXRR(B/X)R) 2 XB, where R is arginine; B is β-alanine; and each X is —C(O)—(CH 2 ) n —NH—, where n is 4-6. The antisense compound is effective to selectively block the sequestration of muscleblind-like 1 protein (MBNL1) and/or CUGBP, in heart and quadricep muscle in a myotonic dystrophy animal model.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/493,140, filed Jun. 26, 2009, which is a continuation-in-part of U.S.patent application Ser. No. 12/217,040, filed Jun. 30, 2008, whichclaims the benefit of U.S. Provisional Application No. 60/937,725, filedJun. 29, 2007; these applications are incorporated herein by referencein their entireties.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 120178_(—)407C2 SEQUENCE_LISTING.txt. The textfile is 18 KB, was created on Aug. 26, 2011, and is being submittedelectronically via EFS-Web.

FIELD OF THE INVENTION

The invention relates to an antisense compound and method for treatingmyotonic dystrophy DM1 and DM2.

REFERENCES

Abes, S., H. M. Moulton et al. (2006). “Vectorization of morpholinooligomers by the (R-Ahx-R)₄ peptide allows efficient splicing correctionin the absence of endosomolytic agents.” J Control Release 116(3):304-13.

Arap, W. et al. (2004). “Human and mouse targeting peptides identifiedby phage display.” U.S. Appn. Pubn. No. 20040170955.

Behlke, M. A. (2006). “Progress towards in vivo use of siRNAs.” Mol Ther13(4): 644-70.

Alter, J., F. Lou et al. (2006). “Systemic delivery of morpholinooligonucleotide restores dystrophin expression bodywide and improvesdystrophic pathology.” Nat Med 12(2): 175-7.

Chen, C. P., L. R. Zhang et al. (2003). “A concise method for thepreparation of peptide and arginine-rich peptide-conjugated antisenseoligonucleotide.” Bioconjug Chem 14(3): 532-8.

Gebski, B. L., C. J. Mann et al. (2003). “Morpholino antisenseoligonucleotide induced dystrophin exon 23 skipping in mdx mousemuscle.” Hum Mol Genet 12(15): 1801-11.

Jearawiriyapaisarn, Moulton et al. (2008). “Sustained DystrophinExpression Induced by Peptide-conjugated Morpholino Oligomers in theMuscles of mdx Mice.” Mol Therapy, Jun. 10, 2008 (advance onlinepublication).

Kang, S. H., M. J. Cho et al. (1998). “Up-regulation of luciferase geneexpression with antisense oligonucleotides: implications andapplications in functional assay development.” Biochemistry 37(18):6235-9.

Kolonin, M. G., J. Sun et al. (2006). “Synchronous selection of homingpeptides for multiple tissues by in vivo phage display.” FASEB J 20(7):979-81.

Meade, B. R. and S. F. Dowdy (2007). “Exogenous siRNA delivery usingpeptide transduction domains/cell penetrating peptides.” Adv Drug DelivRev 59(2-3): 134-40.

Richard, J. P., K. Melikov et al. (2003). “Cell-penetrating peptides. Areevaluation of the mechanism of cellular uptake.” J Biol Chem 278(1):585-90.

Rothbard, J. B., E. Kreider et al. (2002). “Arginine-rich moleculartransporters for drug delivery: role of backbone spacing in cellularuptake.” J Med Chem 45(17): 3612-8.

Samoylova, T. I. and B. F. Smith (1999). “Elucidation of muscle-bindingpeptides by phage display screening.” Muscle Nerve 22(4): 460-6.

Sazani, P., F. Gemignani et al. (2002). “Systemically deliveredantisense oligomers upregulate gene expression in mouse tissues.” NatBiotechnol 20(12): 1228-33.

Sontheimer, E. J. (2005). “Assembly and function of RNA silencingcomplexes.” Nat Rev Mol Cell Biol 6(2): 127-38.

Vodyanoy, V. et al. (2003). “Ligand sensor devices and uses thereof.”U.S. Appn. Pubn. No. 20030640466.

Wu, R. P., D. S. Youngblood et al. (2007). “Cell-penetrating peptides astransporters for morpholino oligomers: effects of amino acid compositionon intracellular delivery and cytotoxicity.” Nucleic Acids Res.35(15):5182-91. (Epub 2007 Aug. 1.)

Youngblood, D. S., S. A. Hatlevig et al. (2007). “Stability ofcell-penetrating peptide-morpholino oligomer conjugates in human serumand in cells.” Bioconjug Chem 18(1): 50-60.

BACKGROUND OF THE INVENTION

The practical utility of many drugs having potentially useful biologicalactivity is often hindered by problems in delivering such drugs to theirtargets. The delivery of drugs and other compounds into cells generallyoccurs from an aqueous cellular environment and entails penetration of alipophilic cell membrane to gain cell entry.

Oligonucleotides and their analogs are one class of potentially usefuldrugs whose practical utility has been impeded due to insufficientcellular uptake, and it has been proposed heretofore to enhance uptakeof oligonucleotides through conjugation of arginine-rich peptidescontaining non-a amino acids (see, for example, Chen, Zhang et al. 2003;Abes, Moulton et al. 2006; Youngblood, Hatlevig et al. 2007; and Wu etal. 2007). The use of arginine-rich peptides has been reported for thetransport of therapeutic drugs, more generally (see, for example,Rothbard, Kreider et al. 2002).

Studies by the inventors and others (Chen, Zhang et al. 2003; Abes,Moulton et al. 2006; Youngblood, Hatlevig et al. 2007) have establishedthat incorporation of unnatural amino acids can confer enhancedstability to peptide carriers and enhanced antisense activity toconjugated oligomers, and therefore improve the potential of CPPs (cellpenetrating peptides) to deliver therapeutic macromolecules.

Parent U.S. application Ser. No. 12/217,040 discloses studies showingthat two of the CPPs reported in the application are effective inselectively targeting oligonucleotides to muscle tissue, particularlyheart muscle, but also including quadricep (skeletal) muscle. These twopeptides have the generic sequence (RXRR(B/X)R)₂XB, where R is arginine;B is β-alanine; and each X is —C(O)—(CH₂)_(n)—NH—, where n is 4-6.Additional studies reported herein confirm the ability of these two CPPsto enhance the uptake and functioning in muscles of oligonucleotideantisense compounds conjugated to one of the CCPs.

The parent application disclosed and claimed the use of these two CPPsfor targeting antisense oligonucleotides to muscle tissue, in treatingcertain muscle pathologies. For example, in treating Duchenne musculardystrophy (DMD), an oligonucleotide designed to promote exon skipping ina mutated dystrophin pre-mRNA (for purposes of restoring the properreading frame in a mutated dystrophin mRNA), is conjugated to one of theCPPs, for enhanced uptake and functioning the oligonucleotide in muscletissue, including both skeletal and heart muscle. In treating DMD, it isadvantageous to effectively target and treat heart muscle, sinceimprovement in skeletal muscle function alone can place aDMD-compromised heart under even greater stress.

The present invention applies this strategy additionally to thetreatment of myotonic dystrophy MD1 and MD2 in muscle tissue, includingskeletal and heart muscle tissue. This condition is associated with longpolyCUG (MD1) and polyCCUG (MD2) repeats in the 3′-UTR regions of thetranscript dystrophia myotonica protein kinase (DMPK). While normalindividuals have as many as 30 CTG repeats, DM1 patients carry a largernumber of repeats ranging from 50 to thousands. The severity of thedisease and the age of onset correlates with the number of repeats.Patients with adult onsets show milder symptoms and have less than 100repeats, juvenile onset DM1 patients carry as many as 500 repeats andcongenital cases usually have around a thousand CTG repeats. Theexpanded transcripts containing CUG repeats form a secondary structure,accumulate in the nucleus in the form of nuclear foci and sequesterRNA-binding proteins (RNA-BP). Several RNA-BP have been implicated inthe disease, including muscleblind-like (MBNL) proteins and CUG-bindingprotein (CUGBP). MBNL proteins are homologous to Drosophila muscleblind(Mbl) proteins necessary for photoreceptor and muscle differentiation.MBNL and CUGBP have been identified as antagonistic splicing regulatorsof transcripts affected in DM1 such as cardiac troponin T (cTNT),insulin receptor (IR) and muscle-specific chloride channel (ClC-1).

MD1 and MD2 are associated with a variety of serious pathologiesincluding muscle abnormalities and weakness, and in the heart,conduction abnormalities.

SUMMARY OF THE INVENTION

The invention includes, in one aspect, an antisense compound for use intreating myotonic dystrophy DM1 or DM2. The compound is composed of anantisense oligonucleotide having 8-30 bases, with at least 8 contiguousbases being complementary to polyCUG or polyCCUG repeats, e.g., SEQ IDNOS: 83 and 84, respectively, in the 3′UTR region of dystrophiamyotonica protein kinase (DMPK) mRNA in DM1 or DM2, respectively, andconjugated to the oligonucleotide, a cell-penetrating peptide having thesequence (RXRR(B/X)R)₂XB, where R is arginine; B is β-alanine; and eachX is —C(O)—(CH₂)_(n)—NH—, where n is 4-6. The compound is effective toselectively block the sequestration of muscleblind-like 1 protein(MBNL1) and/or CUGBP in heart and quadricep muscle in a myotonicdystrophy animal model. Exemplary oligonucleotide sequences for MD1include SEQ ID NOS: 44-47. An exemplary oligonucleotide sequence for MD2includes SEQ ID NO: 48.

In one general embodiment, the cell penetrating peptide has the form(RXRRBR)₂XB (SEQ ID NO: 19) and X is —C(O)—(CH₂)₆—NH—, and theoligonucleotide is a phosphorodiamidate oligonucleotide (PMO) havingbetween 12-30 bases, and at least 12 contiguous bases that arecomplementary to (i) the polyCUG repeats in the 3′UTR region ofdystrophia myotonica protein kinase (DMPK) mRNA in DM1, or (ii) thepolyCCUG repeats in the 3′UTR region of dystrophia myotonica proteinkinase (DMPK) mRNA in DM2.

In another general embodiment, the cell penetrating peptide has the form(RXRRXR)₂XB (SEQ ID NO: 11) and X is —C(O)—(CH₂)₆—NH—, and theoligonucleotide is a phosphorodiamidate oligonucleotide (PMO) havingbetween 12-30 bases, and at least 12 contiguous bases that arecomplementary to (i) the polyCUG repeats in the 3′UTR region ofdystrophia myotonica protein kinase (DMPK) mRNA in DM1, or (ii) thepolyCCUG repeats in the 3′UTR region of dystrophia myotonica proteinkinase (DMPK) mRNA in DM2.

The compound may further include a homing peptide which is selective formuscle tissue, conjugated to the cell-penetrating peptide. Exemplaryhoming peptides have one of the sequences identified as SEQ ID NOS:51-60, particularly SEQ ID NPO:51. The compound preferably has the formcell-penetrating peptide—homing peptide—antisense oligomer.

In another aspect, the invention includes a method of targeting asystemically administered antisense oligonucleotide to heart tissue in amammalian subject, where the oligonucleotide is directed against thepolyCUG or polyCCUG repeats in the 3′UTR region of dystrophia myotonicaprotein kinase (DMPK) mRNA in DM1 or DM2, respectively. The methodincludes conjugating to the oligonucleotide, a cell-penetrating peptidehaving the sequence (RXRR(B/X)R)₂XB, where R is arginine; B isβ-alanine; and each X is independently a neutral linear amino acid—C(O)—(CH₂)_(n)—NH—, where n is 4-6. In various general embodiments,preferred compounds formed by the conjugation are as given above.

In still another aspect, the invention includes a method of treatingmytonic dystrophy DM1 or DM2 in a mammalian subject. The methodincludes, administering to the subject, an antisense compound comprisingan antisense oligonucleotide having 8-30 bases, with at least 8contiguous bases being complementary to the polyCUG or polyCCUG repeatsin the 3′UTR region of dystrophia myotonica protein kinase (DMPK) mRNAin DM1 or DM2, respectively, and conjugated to the oligonucleotide, acell-penetrating peptide having the sequence (RXRR(B/X)R)₂XB, where R isarginine; B is β-alanine; and each X is —C(O)—(CH₂)_(n)—NH—, where n is4-6, and repeating said administering at least once every week to onceevery 3 months.

The cell penetrating peptide may have the form (RXRRBR)₂XB, or(RXRRXR)₂XB, where X is —C(O)—(CH₂)₆—NH—, and the oligonucleotide may bea phosphorodiamidate oligonucleotide (PMO) having between 12-30 bases,and at least 12 contiguous bases that are complementary to the polyCUGor polyCCUG repeats in the 3′UTR region of dystrophia myotonica proteinkinase (DMPK) mRNA in DM1 or DM2, respectively.

The compound may be administered by intravenous or subcutaneousinjection to the subject, at a dose between 1-5 mg/kg body weightantisense compound, and the administering step may be continued atregular intervals of every two weeks to three months. The subject may bemonitored during the treatment for improvement in muscle performance,heart conduction properties, and/or for a reduction in serum creatinekinase.

In still other aspects, the inventions includes methods and compoundsfor treating DMD and muscle atrophy, in accordance with methods andcompositions detailed below.

These and other objects and features of the invention will become morefully apparent when the following detailed description of the inventionis read in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-C show exemplary structures of a phosphorodiamidate-linkedmorpholino oligomer (PMO), a peptide-conjugated PMO (PPMO), and apeptide-conjugated PMO having cationic intersubunit linkages (PPMO+),respectively. (Though multiple cationic linkage types are illustrated inFIG. 1C, a PMO+ or PPMO+ oligomer will typically include just one typeof cationic linkage.)

FIGS. 2A-B show the cellular uptake of conjugates of various cellpenetrating peptides (CPPs) with carboxyfluorescein-labeled morpholinooligomers (PMOF) in pLuc705 cells.

FIGS. 3A-D show the nuclear antisense activity of carrier peptide-PMOconjugates in the presence or absence of 10% serum (A-C) or in thepresence of up to 60% serum (D).

FIG. 4 shows the nuclear antisense activity of carrier peptide-PMOconjugates as a function of the number and position of 6-aminohexanoicacid (Ahx) residues in the peptides.

The peptides 0, 2, 3a, 3b, 3c, 3d, 4a, 4b, 4c, 5 and 8, corresponding tothe number of X residues in the peptide, are shown in Table 1 as SEQ IDNOs: 14, 20, 22, 19, 21, 25, 24, 23, 26, 11 and 3, respectively.

FIGS. 5A-F show the relative toxicity of carrier peptide-PMO conjugates,as measured by MTT assay.

FIGS. 6A-D show the relative toxicity of carrier peptide-PMO conjugatesas measured by PI exclusion (A-C) and hemolysis (D) assays.

FIGS. 7A-P show the splice-correction activity in various organs fromEGFP-654 transgenic mice treated with various EGFP-654-targeted cellpenetrating peptide-PMO conjugates (SEQ ID NOs: 2, 6, 11, 13, 14 and19-27) as measured in diaphragm (FIG. 7A), mammalian gland (FIG. 7B),ovary and prostate (FIG. 7C), brain (FIG. 7D), kidney (FIG. 7E), bonemarrow (FIG. 7F), colon (FIG. 7G), muscle (FIG. 7H), skin (FIG. 7I),spleen (FIG. 7J), stomach (FIG. 7K), thymus (FIG. 7L), heart (FIG. 7M),lungs (FIG. 7N), small intestine (FIG. 7O), and liver (FIG. 7P).

FIG. 8 shows the effect of conjugating an antisense oligomer with amuscle-specific cell penetrating peptide (SEQ ID NO: 19; referred toherein as peptide “B” and also designated CP06062) in combination with amuscle specific homing peptide (MSP), as measured by restoration offull-length dystrophin in the MDX mouse model.

FIG. 9 shows a comparison of dystrophin induction in TA muscles withM23d PMO (SEQ ID NO: 37) and M23d-CP06062 PPMO (SEQ ID NO: 37 conjugatedto SEQ ID NO: 19) by intramuscular injections. The muscles of adult MDXmice were injected with 2 micrograms of each antisense composition andexamined by immunohistochemistry with rabbit polyclonal antibody P7against dystrophin 2 weeks after the injection. Muscle from normal C57BLmouse (b) and mdx mouse injected with 2 micrograms M23d PMO (c), 2micrograms M23d-CP06062 PPMO (d), and 2 micrograms scrambled PPMO (e).Eighty-five percent of the muscle fibers were induced to expressdystrophin after M23d-CP06062 PPMO treatment (d), compared with only 14%of fibers after M23d PMO treatment (c). Only a few revertant fibers weredetected in the muscle treated with the scrambled PPMO (e). Blue nuclearstaining with DAPI. (Scale bar, 50 m.)

FIG. 10 shows the structures of PMO and PPMO (A) and restoration ofdystrophin in muscles of mdx mice (aged 4-5 weeks) after a single i.v.injection of 30 mg/kg of M23d-CP06062 PPMO. The muscles were examined 2weeks after injection. (B-D) Detection of dystrophin byimmunohistochemistry with rabbit polyclonal antibody P7 againstdystrophin. Blue nuclear staining with DAPI. (Scale bar, 100 μM.)Muscles from normal C57BL mice (B), scrambled PPMO-treated mdx mice (C),and M23d-CP06062 PPMO-treated mdx mice (D). Dystrophin was homogenouslyexpressed in all muscle fibers from the M23d-CP06062 PPMO-treated mice.(E) Western blot demonstrated dystrophin in all muscles detected withthe NCL-DYS1 monoclonal antibody. C57-TA, tibialis anterior muscle fromnormal C57BL; Gastro, gastrocnemius; Control-TA, muscle from thescrambled PPMO-treated mdx mouse. (F) Western blot for α-actin asprotein loading control. (G) Detection of exon 23-skipped dystrophinmRNA by RT-PCR. The upper 1,093-bp bands (indicated by E22-E23-E24)correspond to the normal mRNA, and the lower 880-bp bands (indicated byE22-E24) correspond to the mRNA with exon 23 skipped. Sequencing of the880-bp RT-PCR product confirmed the skipping of the exon 23 (H).

FIG. 11 shows the restoration of dystrophin in bodywide muscles of mdxmice (age 4-5 weeks) after six i.v. injections of 30 mg/kg ofM23d-CP06062 PPMO at biweekly intervals. Muscles were examined 2 weeksafter the last injection. Muscles from normal C57BL mice (A), scrambledPPMO-treated mdx mice (B), and M23d-CP06062 PPMO-treated mdx mice (C).Blue nuclear staining with DAPI. Dystrophin was expressed homogenouslyin all muscle fibers from the M23d-CP06062 PPMO-treated mdx mice. (Scalebar, 100 μm.) (D) Western blot demonstrated near-normal levels ofdystrophin in all muscles detected with the NCL-DYS1 monoclonalantibody. C57-TA, TA muscle from normal C57BL mouse; Control-TA, TAmuscle from scrambled PPMO-treated mdx mouse; Gastro, gastrocnemius. (E)Western blot for α-actin as protein loading control. (F) Detection ofexon 23 skipping by RT-PCR. Total RNA of 100 ng from each sample wasused for amplification of dystrophin mRNA from exon 20 to exon 26.Control-TA, TA muscle from scrambled PPMO-treated mdx mouse. The upper1,093-bp bands (indicated by E22-E23-E24) correspond to the normaldystrophin mRNA, and the lower 880-bp bands (indicated by E22-E24)correspond to the mRNA with exon 23 skipped.

FIG. 12 shows the restoration of dystrophin in skeletal and smoothmuscles after six cycles of 30-mg/kg M23d-CP06062 PPMO injection. Backthoracic and lumbar muscle (A), digital muscle (B), flexor muscle (C).Smooth muscles (layers between the two arrows) in small intestine ofuntreated mdx mouse (D) and M23d-CP06062 PPMO-treated mdx mouse (E).Arrowhead indicates a revertant fiber. Dystrophin expression in thesmooth muscle of aorta and vena cava (F) and arteries and other vesselsin the lung (G). Dystrophin was detected by immunostaining with rabbitpolyclonal antibody P7. Blue nuclear staining with DAPI. (Scale bars:A-E, 50 μm; F and G, 120 μm.)

FIGS. 13A-13D shows restoration of muscle and cardiac dystrophinexpression in mdx mice. Restoration of dystrophin expression followingsingle 25 mg/kg intravenous injections of the P007-M23d PPMO (SEQ ID NO:37 conjugated to SEQ ID NO: 11) conjugate in adult mdx mice. (A)Immunostaining of muscle tissue cross-sections to detect dystrophinprotein expression and localization in C57BL6 normal control mice (toppanel), untreated mdx mice (middle panel) and P007-M23d PPMO-treated mdxmice (lower panel), showing near normal levels of dystrophin expressionin the treated mice. Muscle tissues analysed were from tibialis anterior(TA), gastrocnemius, quadriceps, biceps, abdominal wall, diaphragm andheart muscles (scale bar=200 microns). (B) RT-PCR to detect exonskipping efficiency at the RNA level demonstrated almost complete exon23 skipping in the peripheral skeletal muscles indicated and 50% exonskipping in heart in treated mdx mice. This is shown by shorterexon-skipped bands (indicated by the boxed numbered 22-24—for exon 23skipping). Truncated transcripts deleted for both exons 22 and 23 werealso seen as indicated by the box 21-24. (C) Western blot for dystrophinexpression in peripheral skeletal muscles showed 100% dystrophinrestoration in all skeletal muscles except the diaphragm and withP007-M23d PPMO conjugate treatment compared with levels found in normalC57BL6 mice. Equal loading of 10 μg protein is shown for each samplewith -actinin expression detected as a loading control. (D) Western blotto detect dystrophin expression in heart tissue from normal C57BL6 heart(20, 10 and 5% of normal levels shown), untreated mdx heart andP007-M23d PPMO treated heart. Data shows dystrophin protein restorationto 15% of normal levels in P007-M23d PPMO treated mdx heart tissue.

DETAILED DESCRIPTION I. Definitions

The terms below, as used herein, have the following meanings, unlessindicated otherwise:

The terms “cell penetrating peptide” or “CPP” are used interchangeablyand refer to cationic cell penetrating peptides, also called transportpeptides, carrier peptides, or peptide transduction domains. Thepeptides, as shown herein, have the capability of inducing cellpenetration within 100% of cells of a given cell culture population andallow macromolecular translocation within multiple tissues in vivo uponsystemic administration.

The terms “antisense oligomer” or “antisense oligonucleotide” or“oligonucleotide” are used interchangeably and refer to a sequence ofcyclic subunits, each bearing a base-pairing moiety, linked byintersubunit linkages that allow the base-pairing moieties to hybridizeto a target sequence in a nucleic acid (typically an RNA) byWatson-Crick base pairing, to form a nucleic acid:oligomer heteroduplexwithin the target sequence. The cyclic subunits are based on ribose oranother pentose sugar or, in a preferred embodiment, a morpholino group(see description of morpholino oligomers below). The oligomer may haveexact or near sequence complementarity to the target sequence;variations in sequence near the termini of an oligomer are generallypreferable to variations in the interior.

In one aspect of the invention, for the treatment of MD1 or MD2, theantisense oligonucleotide is complementary to at least 8, typically 9-12or more, e.g., 15-30 contiguous bases in polyCUG repeats or polyCCUGrepeats within the 3′ UTR regions of the transcript for dystrophiamyotonica protein kinase (DMPK) in muscle cells, and is designed to bindby hybridization to these repeats, blocking binding of splice-associatedproteins, such as one or more muscleblind family proteins, e.g., MBNL1,or CUGBP to the transcript. The oligonucleotide may be said to be“directed to” or “targeted against” 3′UTR polyCUG or polyCCUG repeatswith which it hybridizes. The target sequence may include a polyCUG orpolyCCUG region of at least 8 contiguous bases, preferably at least9-25, and up to 40 bases or more. SEQ ID NOS: 49, 50 define polyCUG andpolyCCUG repeat sequences of 39 and 40 bases, respectively.

In another aspect, for the treatment of DMD, the antisenseoligonucleotide is complementary to at least 8, typically 9-12 or moree.g., 15-30 contiguous bases in a splice junction site or exonrecognition sequence of a dystrophin pre-MRNA, where binding of theoligonucleotide to the target pre-mRNA sequence is effective to promoteskipping of one or more exons in a mutated dystrophin gene, with theresult that the normal reading frame of the processed mRNA is restored.Exemplary targeting sequences includes one from SEQ ID NOS: 28-38.

In still another aspect, for treatment of muscle atrophy, the antisenseoligonucleotide is complementary to at least 8, typically 9-12 or more,e.g., 15-30 bases, in an AUG region of myostatin mRNA or a splicejunction site of a myostatin pre-mRNA, effective to inhibit expressionof a functional myostatin protein in muscle cells. Exemplary targetingsequences includes one from SEQ ID NOS: 39-43.

The terms “morpholino oligomer” or “PMO” (phosphoramidate- orphosphorodiamidate morpholino oligomer) refer to an oligonucleotidecomposed of morpholino subunit structures, where (i) the structures arelinked together by phosphorus-containing linkages, one to three atomslong, preferably two atoms long, and preferably uncharged or cationic,joining the morpholino nitrogen of one subunit to a 5′ exocyclic carbonof an adjacent subunit, and (ii) each morpholino ring bears a purine orpyrimidine base-pairing moiety effective to bind, by base specifichydrogen bonding, to a base in a polynucleotide. See, for example, thestructure in FIG. 1A, which shows a preferred phosphorodiamidate linkagetype. Variations can be made to this linkage as long as they do notinterfere with binding or activity. For example, the oxygen attached tophosphorus may be substituted with sulfur (thiophosphorodiamidate). The5′ oxygen may be substituted with amino or lower alkyl substitutedamino. The pendant nitrogen attached to phosphorus may be unsubstituted,monosubstituted, or disubstituted with (optionally substituted) loweralkyl. See also the discussion of cationic linkages below. The purine orpyrimidine base pairing moiety is typically adenine, cytosine, guanine,uracil, thymine or inosine. The synthesis, structures, and bindingcharacteristics of morpholino oligomers are detailed in U.S. Pat. Nos.5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,521,063, and5,506,337, and PCT Pubn. No. WO 2008036127 (cationic linkages), all ofwhich are incorporated herein by reference.

An “amino acid subunit” or “amino acid residue” can refer to an α-aminoacid residue (—CO—CHR—NH—) or a β- or other amino acid residue(e.g.—CO—(CH₂)_(n)CHR—NH—), where R is a side chain (which may includehydrogen) and n is 1 to 6, preferably 1 to 4.

The term “naturally occurring amino acid” refers to an amino acidpresent in proteins found in nature. The term “non-natural amino acids”refers to those amino acids not present in proteins found in nature,examples include beta-alanine (β-Ala), 6-aminohexanoic acid (Ahx) and6-aminopentanoic acid.

A “marker compound” refers to a detectable compound attached to atransport peptide for evaluation of transport of the resulting conjugateinto a cell. The compound may be visually or spectrophotometricallydetected, e.g. a fluorescent compound or fluorescently labeled compound,which may include a fluorescently labeled oligomer. Preferably, themarker compound is a labeled or unlabeled antisense oligomer. In thiscase, detection of transport involves detection of a product resultingfrom modulation of splicing and/or transcription of a nucleic acid by anantisense oligomeric compound. Exemplary methods, such as a splicecorrection assay or exon skipping assay, are described in Materials andMethods below.

An “effective amount” or “therapeutically effective amount” refers to anamount of therapeutic compound, such as an antisense oligomer,administered to a mammalian subject, either as a single dose or as partof a series of doses, which is effective to produce a desiredtherapeutic effect.

“Treatment” of an individual (e.g. a mammal, such as a human) or a cellis any type of intervention used in an attempt to alter the naturalcourse of the individual or cell. Treatment includes, but is not limitedto, administration of a pharmaceutical composition, and may be performedeither prophylactically or subsequent to the initiation of a pathologicevent or contact with an etiologic agent.

An “antisense compound” or “compound” or “conjugate compound” refers toa compound formed by conjugating the (RXRR(X/B)R)₂XB cell-penetratingpeptides to an oligonucleotide targeted against a muscle-protein gene,e.g., a region of polyCUG or polyCCUG repeats.

“Systemic administration” of a compound refers to administration, suchas intravenous (iv) subcutaneous (subQ), intramuscular (IM), andintraperitoneal (IP) that delivers the compound directly into thebloodstream.

A systemically administered antisense oligonucleotide is targeted toheart muscle tissue by conjugation to the CPP (RXRRBR)₂XB, if thecompound, when administered systemically to a MD1 or MD2 subject inaccordance with the method herein, produces a measurable improvement inheart muscle performance and/or improvement in conduction properties ofthe heart, as measured by known methods.

II. Structural Features of Transport Peptides

The two cell-penetrating peptides employed in the invention are in aclass of a transport peptide having 8 to 30 amino acid residues inlength and consisting of subsequences selected from the group consistingof RXR, RX, RB, and RBR; where R is arginine (which may includeD-arginine, represented in the sequences herein by r), B is β-alanine,and each X is independently —C(O)—(CHR¹)_(n)—NH—, where n is 4-6 andeach R¹ is independently H or methyl, such that at most two R¹'s aremethyl. Preferably, each R¹ is hydrogen. The two peptides have thegeneric formula (RXRR(B/X)R)₂XB, where R is arginine; B is β-alanine;and each X is —C(O)—(CH₂)_(n)—NH—, where n is 4-6, preferably 6, andinclude both (RXRRBR)₂XB (SEQ ID NO: 19) and (RXRRXR)₂XB (SEQ ID NO: 11)and where R is arginine; B is β-alanine; and each X is—C(O)—(CH₂)_(n)—NH—, where n is 4-6. As discussed in Section V below,these two peptides have been discovered to selectively target anoligonucleotide, including a PMO, to muscle tissue, including,importantly, heart muscle tissue.

Table 1 below shows the sequences of various transport peptides in thisclass that were evaluated, in conjugation with suitable antisenseoligonucleotides, for their ability to selectively target varioustissues, including heart and skeletal muscle. The peptides wereevaluated for cellular uptake (Section III), as determined by flowcytometry; for antisense activity (Section IV), as determined by asplice correction assay (Kang, Cho et al. 1998); and for cellulartoxicity, as determined by MTT cell viability, propidium iodide membraneintegrity and hemolysis assays, and microscopic imaging, and theiruptake and functional activity in muscle tissue relative to a variety ofnon-muscle tissue were compared (Section V). As will be seen in SectionIV, the (RXRRXR)₂XB peptide was among the most active in antisenseactivity, as determined by the splice correction assay (the (RXRRBR)₂XBpeptide was not tested in this assay), both in the presence and absenceof added serum. As will seen in Section V, both (RXRR(B/X)R)₂XB peptideswere effective in selectively targeting oligonucleotides to heart andskeletal tissue, while showing relatively low-level targeting to avariety of other tissues, including mammary gland tissue, ovary/prostate(particularly (RXRRXR)₂XB), and brain.

TABLE 1 Cell-Penetrating Peptides SEQ ID Name (Designation) SequenceNO.^(a) Oligoarginines R₈-XB(A; 8) RRRRRRRR-XB 3 r₈-XB rrrrrrrr-XB 4R₉-XB RRRRRRRRR-XB 5 Oligo (RX), (RXR), and (RB) series, includingD-arginine (RX)₈-B RXRXRXRXRXRXRXRX- 6 B (rX)₈-B rXrXrXrXrXrXrXrX-B 7(RX)₇-B RXRXRXRXRXRXRX-B 8 (RX)₅-B RXRXRXRXRX-B 9 (RX)₃-B RXRXRX-B 10(RXR)₄-XB (P007; 5) RXRRXRRXRRXR-XB 11 (rXR)₄-XB rXRrXRrXRrXR-XB 12(rXr)₄-XB (D-P007) rXrrXrrXrrXr-XB 13 (RB)₈-B (0) RBRBRBRBRBRBRBRB- 14 B(rB)₈-B rBrBrBrBrBrBrBrB-B 15 (RB)₇-B RBRBRBRBRBRBRB-B 16 (RB)₅-BRBRBRBRBRB-B 17 (RB)₃-B RBRBRB-B 18(RX), (RXR), (RB), and (RBR) mixed series (RXRRBR)₂-XB (B; 3b;RXRRBRRXRRBR-XB 19 CP06062) (RXR)₃RBR-XB (C; 4c) RXRRXRRXRRBR-XB 26(RB)₅RXRBR-XB (D; 2) RBRBRBRBRBRXRBR- 20 XB (RBRBRBRX)₂-X (E; 3c)RBRBRBRXRBRBRBRX- 21 X X(RB)₃RX(RB)₃R-X (F; 3a) XRBRBRBRXRBRBRBR- 22 X(RBRX)₄-B (G; 4b) RBRXRBRXRBRXRBRX- 23 B (RB)₄(RX)₄-B (H; 4a)RBRBRBRBRXRXRXRX- 24 B RX(RB)₂RX(RB)₃R-X (I; 3d) RXRBRBRXRBRBRBRX - 25 X(RB)₇RX-B RBRBRBRBRBRBRBRX- 27 B ^(a)Sequences assigned to SEQ ID NOs donot include the linkage portion (X, B, or XB).

III. Cellular Uptake of Peptide-Oligomer Conjugates

Cellular uptake of peptide-PMO conjugates, where the PMO was a3′-carboxy fluorescein-tagged PMO (PMOF), was investigated using flowcytometry. A treatment concentration of 2 μM was used because none ofthe conjugates caused any detectable cytotoxicity at this concentration,as demonstrated by MTT and PI uptake assays (below). After incubationwith conjugate, cells were treated with trypsin (Richard, Melikov et al.2003) to remove membrane-bound conjugate. To determine the effect ofserum on cellular uptake of the various conjugates, uptake evaluationassays were carried out in medium containing various concentrations ofserum.

As shown in FIGS. 2A-B, cellular uptake of the conjugates increased withthe number of arginine residues in the transport peptide and generallydecreased with X and/or B residue insertion. For example, theoligoarginine R₉-PMOFconjugate had a mean fluorescence (MF) value of662, nearly 3-fold higher than that of R₈-PMOF. Insertion of an X or Bresidue in the R₈ sequence reduced uptake of the respective conjugates,as shown by MF values for conjugates of R₈ (234), (RX)₈ (42), (RXR)₄(70), and (RB)₈ (60) (FIG. 2A). The number of RX or RB repeats alsoaffected cellular uptake, with conjugates having fewer RX or RB repeatsgenerating lower MF values (FIG. 2B).

While the addition of 10% serum to the medium caused a decrease in theuptake of the oligoarginine R₈- or R₉-PMOF conjugates, it increaseduptake of conjugates containing RX, RB or RXR motifs (FIGS. 2A and 2C).For example, the presence of serum reduced the MF of R₉- and R₈-PMOFfrom 662 and 234 to 354 and 158, respectively, but it increased the MFof (RX)₈-, (RXR)₄-, and (RB)₈-PMOF from 41, 70 and 60 to 92, 92, and111, respectively. These differences were statically significant (FIG.2A). However, higher serum concentrations (30% and 60%) decreased theuptake of both (RXR)₄-PMOF and oligoarginine-PMOF.

Arginine stereochemistry (D vs. L) had little effect on uptake of thepeptide-PMOF conjugates. Uptake as shown by MF values of R₈-, (RB)₈- and(RX)₈-PMOF conjugates was not significantly different from theirrespective D-isomer conjugates, r₈-, (rB)₈- and (rX)₈-PMOF (data notshown).

IV. In Vitro Nuclear Antisense Activity

The effectiveness of the subject peptides in transporting an attachedmolecule to the nucleus of a cell was determined in a splicingcorrection assay (Kang, Cho et al. 1998), where the attached compound isa steric-blocking antisense oligomer (AO), in this case a PMO. Thisassay utilizes the ability of the oligomer to block a splice sitecreated by a mutation in order to restore normal splicing. Specifically,the luciferase coding sequence is interrupted by the human β-globinthalassemic intron 2, which carries a mutated splice site at nucleotide705. HeLa cells were stably transected with the resulting plasmid anddesignated pLuc705 cells. In the pLuc705 system, the oligomer must bepresent in the cell nucleus for splicing correction to occur. Advantagesof this system include the positive readout and high signal-to-noiseratio. With this system, the relative efficiencies of various transportpeptides to deliver an AO with sequence appropriate forsplice-correction to cell nuclei can be easily compared.

As described below, the subject carrier peptide-PMO conjugates displayhigher activity in cell nuclei, and are less affected by serum and morestable in blood, than oligoarginine-PMO conjugates.

Oligoarginine, RX, RXR and RB panels (see Table 1). The peptide-PMOconjugates with the highest nuclear antisense activities in this serieswere found to be (RXR)₄- and (RX)₈-PMO (where, as noted above, R isarginine, and X in these peptides is 6-aminohexanoic acid). FIGS. 3A and3B show luciferase activity normalized to protein of cells treated withvarious conjugates at 1 μM and 5 μM for 24 hr. At both concentrations,(RX)₈- and (RXR)₄-PMO were more effective than the other conjugatestested, with the difference more prominent in serum-containing medium at1 μM than at 5 μM. Cells treated with 1 μM of either conjugate exhibitedluciferase activity at a level 10-15 fold over background, while theremaining conjugates yielded about a 2-4 fold increase over background(FIG. 3A). At 5 μM, all conjugates generated higher luciferase activitythan at 1 μM, with (RX)₈-PMO and (RXR)₄-PMO again the most effective,followed by (RB)₈-PMO (FIG. 3B).

FIG. 3C shows that, at 10 μM, the activity of RX or RB conjugatesdecreased as the number of RX or RB repeats (i.e. length) in thetransport peptide decreased. The peptides with three or five RX or RBrepeats generated much lower luciferase activity than those with sevenor eight repeats.

Number and position of X residues. In order to investigate the effect ofthe number and position of X residues on the activity of conjugates,eleven peptide-PMO conjugates, where the peptide component contained 0,2, 3, 4, 5, or 8 X (6-aminohexanoic acid) residues, were compared (SEQID NOs: 14, 20, 22, 19, 21, 25, 24, 23, 26, 11 and 3 as shown in Table1). The data (shown as lucifrase activity in the assay described above)is presented in FIG. 4.

Generally, peptides containing a higher number of X residues had highertransport activities. At 2 μM, (RX)₈-PMO (eight X residues) had thehighest activity, followed by (RXR)₄-PMO (five X residues), and theconjugates with fewer X residues had lower activities.

At 5 μM, three conjugates containing three (I; SEQ ID NO: 25), four (C;SEQ ID NO: 26) and eight ((RX)₈) 6-aminohexanoic acid residues had thehighest activities, suggesting that the position of X residues affectsactivity.

Serum effect on activity. The effect of serum on the antisense activityof the conjugates was dependent on the peptide sequences, as shown inFIGS. 3A-3D. Addition of 10% serum to the medium decreased the activityof oligoarginine-PMO conjugates (R₈-PMO and R₉-PMO) but increasedactivity of conjugates containing RXR, RX and RB repeats. The additionof 10% serum nearly doubled the luciferase activity of (RXR)₄-, (RX)₈-and (RB)₈-PMO at 5 μM (FIG. 3B). This effect was further investigatedfor (RXR)₄-PMO up to 60% serum (see FIG. 3D). While the activity almostdoubled as the serum concentration increased from 0% to 10%, itgradually decreased as the serum concentration increased to 60%, atwhich activity was similar to that in 0% serum (which was stillsignificantly above background). This “up and down” profile was alsoobserved with the 1 μM (RXR)₄-PMO treatment. Unlike (RXR)₄-PMO, theluciferase activity of R₈-PMO or R₉-PMO consistently decreased as theserum concentration increased, with an approximately 30% reduction in10% serum and no activity in 60% serum (FIG. 3D). R₈-PMO or R₉-PMO didnot display any detectable activity at 1 μM, regardless of the serumconcentration (FIG. 3A).

V. Tissue Selectivity for In Vivo Nuclear Antisense Activity

Various transport peptides were conjugated to PMO, and the resultingconjugates (P-PMOs) were tested for their ability to transport the PMOinto various tissues, in accordance with the invention,as describedfurther in Materials and Methods, below. Briefly, conjugates wereadministered for four consecutive days. The in vivo uptake of the P-PMOswas determined by targeting the PMO (SEQ ID NO: 1) to an aberrantlyspliced mutated intron in the EGFP-654 gene in an EGFP-654 transgenicmouse model (Sazani, Gemignani et al. 2002). In this model, cellularuptake of the EGFP-654 targeted P-PMOs can be evaluated by RT-PCRdetection of restored EGFP-654 mRNA splice product and functionallyrestored EGFP in tissues harvested after IP administration of P-PMO.

As shown in FIGS. 7A-P, P-PMOs containing various transport peptidesdisplayed selective uptake by specific tissues. In particular, aconjugate containing the transport peptide (RXRRBR)₂-XB (SEQ ID NO: 19)displayed selective uptake into heart, and skeletal muscle, as well aslungs, lungs, small intestine, colon, stomach, skin, and bone marrow,while uptake into other organs, including mammary gland, ovary/prostateand brain, was greatly reduced in comparison. Similarly, the conjugatecontaining the peptide or (RXRRXR)₂-XB (SEQ ID NO: 11) displayedselective uptake into heart, muscle, liver, small intestine, stomach,and mammary gland, while uptake into other organs, including mammarygland, ovary/prostate and brain, was greatly reduced in comparison.

VI. Cellular Toxicity of Carrier Peptide-PMO Conjugates

The cellular toxicity of the various peptide-PMO conjugates wasdetermined by MTT-survival, propidium iodine (PI) exclusion, hemolysisassays, and microscopic imaging. The MTT and PI exclusion assays measuremetabolic activity and membrane integrity of cells, respectively. Thehemolysis assay determines compatibility with blood. Microscopic imageswere used to verify the MTT results and observe the general health ofthe cells. As detailed below, the conjugates generally showed lowtoxicity, with those containing (RX)₈ and (RXR)₄ having the highestlevels of toxicity.

MTT assay (FIGS. 5A-F). pLuc705 cells were treated at concentrationsranging from 2-60 μM for 24 hr. As shown in FIG. 4, all conjugates, withthe exception of those containing (RX)₈ and (RXR)₄, had no toxicity atup to 60 μM. The (RX)₈ and (RXR)₄ conjugates exhibited no toxicity up to10 μM, while at higher concentrations they reduced cell viability in aconcentration-dependent manner, with (RX)₈ being more toxic than (RXR)₄(FIGS. 5C-D).

Replacement of L-arginine with D-arginine in R₈-, (RB)₈- and (RXR)₄-PMOdid not change the viability profiles of these conjugates (FIGS. 5A-C).Surprisingly, the L→D replacement in (RX)₈-PMO decreased the toxicity(FIG. 5D).

The eight conjugates containing peptides with fewer than five X residuesdid not inhibit cell proliferation up to 60 μM (FIG. 5E). Monomers of Ror X, individually or in combination, at 500 μM each, produced noinhibition of cell proliferation (FIG. 5F).

The toxicities of the conjugates (RXR)₄-PMO, RX(RB)₂RX(RB)₃RX-PMO(peptide SEQ ID NO: 25) and (RXR)₃RBR-PMO (peptide SEQ ID NO: 26) werealso evaluated in a human liver HepG2 cells. Of these, only (RXR)₄-PMOcaused dose-dependent inhibition of cell proliferation, while the othertwo conjugates had no toxicity up to 60 μM, the highest concentrationtested in this study.

Microscopic images. Images of cells treated with 60 μM of the conjugatescorrelated well with the MTT cell viability data. Cells treated with(RX)₈-PMO and (RXR)₄-PMO appeared rounded and detached from the culturewell, and appeared to have fewer live cells. Interestingly, cellstreated with (rX)₈-PMO appeared to have normal morphology and celldensity. The replacement of one X of (RXR)₄-PMO with one B reducedtoxicity significantly; i.e., cells treated with (RXR)₃RBR-PMO (peptideSEQ ID NO: 26) had similar density and morphology to the vehicle-treatedcells.

Propidium iodine exclusion assay. The effect of the conjugates onintegrity of cell membranes was investigated by a propidium iodine (PI)exclusion assay. PI can permeate only unhealthy/damaged membranes;therefore, positive PI fluorescence indicates compromised cellmembranes. Only (RXR)₄-PMO and (RX)₈-PMO conjugates were found tosignificantly affect membrane integrity at higher concentrations (up to60 μM tested).

FIG. 6A shows the histograms of pLuc705 cells treated with (RXR)₄-PMO at60 μM for 0.5, 5 and 24 hr. The PI positive (PI+) region was defined bythe cells permeabilized with ethanol (positive control) as indicated bythe gate in the histogram. The PI histogram shifts from the PI-negativeregion to PI-positive region in the longer incubations, indicating theconjugate caused membrane leakage in a time-dependent manner. The 0.5hr- and 5 hr-treatments caused a slight shift towards the PI+ region,while the 24 hr-treatment produced a distinct peak which corresponds to57% of cells that were in the PI+ region.

FIG. 6B shows the histograms of cells treated with (RXR)₄-PMO atconcentrations of 2, 10, 20, 40 and 60 μM for 24 hr. There was nosignificant PI uptake at concentrations up to 20 μM. At higherconcentrations, the PI+ population appeared, and the percentage of PI+cells increased as the treatment concentration increased, indicatingthat there were more leaking cells at the higher treatmentconcentration. Similar concentration- and time-dependent PI uptakeprofiles were observed for (RX)₈-PMO, but not for (RB)₈-PMO and theremaining conjugates. Addition of 10% serum to the treatment mediumsignificantly reduced membrane toxicity for the (RXR)₄- (FIG. 6C) and(RX)₈-PMO conjugates.

Hemolysis assay. The (RXR)₄- and (RX)₈-PMO conjugates were tested in ahemolysis assay and found to be compatible with red blood cells. Freshrat red blood cells were treated with the conjugates at 60 μM, PBS(background) or 0.005% TX-100 (positive control). The supernatants ofconjugate- and PBS-treated samples had small and similar amounts of freehemoglobin released, far lower than that of the TX-100-treated samples(FIG. 6D).

Animal studies on compounds containing both (RXRRXR)₂XB and (RXRRBR)₂XBpeptides show that the conjugate compounds are well tolerated, withlittle or no observable toxicity effects at therapeutically effectivedoses, and with the (RXRRBR)₂XB peptide showing lower toxicity than the(RXRRXR)₂XB peptide at elevated compound doses.

VII. Therapeutic Applications

The carrier peptides and conjugate compounds of the present inventionare useful for targeting and delivering an antisense oligomer, such as aPMO, across both the cell and nuclear membranes to the nucleus of musclecells in skeletal and heart muscle tissue, by exposing the cell to aconjugate comprising the oligomer covalently linked to a carrier peptideas described above.

(A1) Treatment of Duchenne muscular dystrophy. In one embodiment, anantisense oligomer conjugated to a muscle-specific carrier peptide asdescribed herein is used in an improved method for treating Duchennemuscular dystrophy (DMD). Mutations in the human dystrophin gene can beremoved from the processed mRNA by antisense oligomers that cause exonskipping of the exon containing the mutation. The resulting processeddystrophin mRNA can encode a functional dystrophin protein. An exemplaryantisense oligomer targeted to exon 51 of the human dystrophin gene (SEQID NO: 38) induces skipping of exon 51. Other suitable antisenseoligomers include those having SEQ ID NOs: 28-36 for human treatment andSEQ ID NO:37 used in the mouse MDX model.

This therapeutic strategy can benefit greatly from the use ofmuscle-specific carrier peptides (RXRR(B/X)R)₂XB, as detailed inExamples 2-4 below. Treatment of the MDX mouse using the M23d-CP06062PPMO (SEQ ID NO 37 conjugated to SEQ ID NO: 19) compound demonstratedsuperior delivery of the PPMO to all muscle tissues including cardiactissues as described in Example 3 and shown in FIGS. 9-12.

Treatment of DMD, in accordance with the invention, comprises:

(i) administering to the subject, an antisense compound comprising atherapeutic oligonucleotide of the type indicated above for restoringthe normal reading frame in a mutated dystrophin mRNA, and conjugated tothe oligonucleotide, a cell-penetrating peptide having the sequence(RXRR(B/X)R)₂XB, where R is arginine; B is β-alanine; and each X is—C(O)—(CH₂)₆—NH—, where n is 4-6, and

(ii) repeating compound administration at least once every one week toonce every three months or longer.

Exemplary oligonucleotide sequences include SEQ ID NOS: 28-38. Thecompound is preferably administered by intravenous or subcutaneousinjection to the subject, at a dose between 1-5 mg/kg body weightantisense compound, at a dosing schedule of once a month to once every2-3 months. For subQ administration, the dose required may be roughlytwice that for IV administration. During the course of treatment, thepatient is monitored for improvement or stabilization of muscleperformance and heart function, according to established procedures.Because muscular dystrophy is a chronic disease, the treatment methodwill be applied over the subject's lifetime, with dose adjustments beingmade during the treatment period to achieve a desired level of musclefunction and to accommodate patient growth.

As can be seen from the findings in Examples 3 and 4, the treatmentmethod offers a number of important advantages over earlier proposedantisense methods of treating DMD. First, targeting, uptake andantisense activity of the antisense compound into and in both skeletaland heart muscle is efficient, leading to a high percentage of musclefibers in skeletal and heart muscle showing dystrophin expression. Thisallows effective treatment with relatively modest compound doses, e.g.,in the range 1-5 mg/kg subject weight. Secondly, little or no compoundtoxicity is observed, as evidenced by no observable increases in muscledamage, inflammatory cellular infiltrates, or necrotic fibers wereobserved microscopically in the muscles injected with any of the PPMOsand PMO. Finally, the effect of a single dose may be effective for up tothree months or more, allowing the patient to be effectively treated bydosing at intervals of no less than one month, and up to 3 months ormore between successive treatments.

(A2) Treatment of Myotonic Dystrophy. As the name of the disorderimplies the characteristic clinical manifestation in DM is myotonia(muscle hyperexcitability) and muscle degeneration. Affected individualswill also develop insulin resistance, cataracts, heart conductiondefects, testicular atrophy, hypogammaglobulinemia and sleep disorders.Symptoms of DM can manifest in the adult or in childhood. The childhoodonset form of the disease is often associated with mental retardation.In addition, there is a form of the disease referred to as congenitalmyotonic dystrophy. This latter form of the disease is frequently fataland is seen almost exclusively in children born of mothers whothemselves are mildly affected by the disease. In congenital DM thefacial manifestations are distinctive due to bilateral facial palsy andmarked jaw weakness. Many infants with congenital DM die due torespiratory insufficiency before a proper diagnosis of the disease ismade.

DM1 initially involves the distal muscles of the extremities and only asthe disease progresses do proximal muscles become affected. In addition,muscles of the head and neck are affected early in the course of thedisease. Weakness in eyelid closure, limited extraocular movement andptosis results from involvement of the extraocular muscles. Manyindividuals with DM1 exhibit a characteristic “haggard” appearance thatis the result of atrophy of the masseters (large muscles that raise andlower the jaw), sternocleidomastoids (large, thick muscles that passobliquely across each side of the neck and contribute to arm movement)and the temporalis muscle (muscle involved in chewing).

Treatment of MD1 comprises or MD2, in accordance with the invention,:

(i) administering to the subject, an antisense compound comprising anantisense oligonucleotide having 8-30 bases, with at least 8 contiguousbases being complementary to the polyCUG or polyCCUG repeats in the3′UTR region of dystrophia myotonica protein kinase (DMPK) mRNA in DM1or DM2, respectively, and conjugated to the oligonucleotide, acell-penetrating peptide having the sequence (RXRR(B/X)R)₂XB, where R isarginine; B is β-alanine; and each X is —C(O)—(CH₂)_(n)—NH—, where n is4-6, and

(ii) repeating the compound administration at least once every one weekto once every three months or longer.

As with DMD treatment, the compound is preferably administered byintravenous or subcutaneous injection to the subject, at a dose between1-5 mg/kg body weight antisense compound, at a dosing schedule of once amonth to once every 2-3 months. For subQ administration, the doserequired may be roughly twice that for IV administration. During thecourse of treatment, the patient is monitored for improvement orstabilization of muscle performance, improvement in heart conductionproperties and/or reduction in serum reduction in serum creatine kinase.Because myotonic dystrophy is a chronic disease, the treatment methodwill be applied over the subject's lifetime, with dose adjustments beingmade during the treatment period to achieve a desired level of musclefunction and to accommodate patient growth.

As discussed above for DMD treatment, the treatment method offers anumber of important advantages over earlier proposed antisense methodsof treating MD1 or MD2. First, targeting, uptake and antisense activityof the antisense compound into and in both skeletal and heart muscle isefficient, as demonstrated for antisense oligonucleotide targetedagainst muscle dystrophin protein. This allows effective treatment withrelatively modest compound doses, e.g., in the range 1-5 mg/kg subjectweight. Secondly, little or no compound toxicity is observed, asevidenced by no observable increases in muscle damage, inflammatorycellular infiltrates, or necrotic fibers were observed microscopicallyin the muscles injected with any of the PPMOs and PMO. Finally, as inthe DMD treatment method, the effect of a single dose may be effectivefor up to three months or more, allowing the patient to be effectivelytreated by dosing at intervals of no less than one month, and up to 3months or more between successive treatments.

(A3) Treatment of muscle atrophy. In another embodiment, an antisenseoligomer as described herein can be used in a method for treating lossof skeletal muscle mass in a human subject. The steps in the methodentail:

(a) measuring blood or tissue levels of myostatin in the subject,

(b) administering to the subject, a myostatin-expression-inhibitingamount of an oligomer as described herein, conjugated to(RXRR(B/X)R)₂XB, where R is arginine; B is β-alanine; and each X is—C(O)—(CH₂)_(n)—NH—, where n is 4-6, and having a base sequenceeffective to hybridize to an expression-sensitive region of processed orpreprocessed human myostatin RNA transcript;

(c) by this administering, forming within target muscle cells in thesubject, a base-paired heteroduplex structure composed of humanmyostatin RNA transcript and the antisense compound and having a Tm ofdissociation of at least 45° C., thereby inhibiting expression ofmyostatin in said cells;

(d) at a selected time following administering the antisense compound,measuring a blood or tissue level of myostatin in the subject; and

(e) repeating the administering, using the myostatin levels measured in(d) to adjust the dose or dosing schedule of the amount of antisensecompound administered, if necessary, so as to reduce measured levels ofmyostatin over those initially measured and maintain such levels ofmyostatin measured in step (d) within a range determined for normal,healthy individuals.

Where the antisense oligomer is effective to hybridize to a splice siteof preprocessed human myostatin transcript, it has a base sequence thatis complementary to at least 12 contiguous bases of a splice site in apreprocessed human myostatin transcript, and formation of theheteroduplex in step (c) is effective to block processing of apreprocessed myostatin transcript to produce a full-length, processedmyostatin transcript. Exemplary antisense sequences are those identifiedby SEQ ID NOs: 39-43.

Compound doses and dose schedules are similar to those described abovefor DMD treatment and MD treatment, as are the advantages achievable bythe treatment method.

VIII. Combination with Homing Peptides

The oligonucleotide-(RXRR(B/X)R)₂XB conjugate compounds of the inventionmay be used in conjunction with homing peptides selective for the targettissue, to further enhance muscle-specific delivery. An example of thisapproach can be found in the application of muscle-binding peptides(Samoylova and Smith, 1999; Vodyanoy et al., U.S. Appn. Pubn. No.20030640466) coupled to antisense oligomers designed to be therapeutictreatments for Duchenne muscular dystrophy (DMD) (Gebski, Mann et al.2003; Alter, Lou et al. 2006) (PCT Pubn. No. WO2006000057). Theheptapeptide sequence ASSLNIA has enhanced in vivo skeletal and cardiacmuscle binding properties, as described by Samoylova and Smith. As afurther example, a pancreas-homing peptide, CRVASVLPC, mimics thenatural prolactin receptor ligand (Kolonin, Sun et al. 2006).

An exemplary dual peptide molecule has a cell penetrating peptide to oneterminus, e.g. at the 5′ end of the antisense oligomer, as describedherein, and a homing peptide coupled to the other terminus, i.e. the 3′terminus. The homing peptide localizes the peptide-conjugated PMO to thetarget tissue, where the cell-penetrating peptide moiety effectstransport into the cells of the tissue.

Alternatively, a preferred exemplary dual peptide molecule would haveboth a homing peptide (HP) and cell-penetrating peptide (CPP) conjugatedto one end, e.g. the 5′ terminus of the antisense oligomer, in either aHP-CPP-PMO configuration or, more preferably, a CPP-HP-PMOconfiguration.

For example, a PMO designed to induce therapeutic exon skipping of thedystrophin gene, as described by Wilton et al. (PCT PublicationWO2006/000057), conjugated at the 3′ terminus to the muscle-bindingpeptide ASSLNIA, and further coupled at the 5′ terminus to a cellpenetrating peptide of the present invention, preferably having enhancedselectivity for muscle tissue, will provide enhanced therapeuticpotential in the treatment of DMD. This is exemplified in Example 2,below.

TABLE 2 Examples of Muscle-specific Homing Peptides (HP) SEQPeptide Sequence (NH₂ to ID Target Tissue COOH) NO. Skeletal Muscle-ASSLNIA 51 SMP1 SMP2 SLGSFP 52 SMP3 SGASAV 53 SMP4 GRSGAR 54 SMP5TARGEHKEEELI 55 Cardiac Muscle- WLSEAGPVVTVRALRGTGSW 56 CMP1 CMP2VTVRALRGTSW 57 CMP3 VVTVRALRGTGSW 58 CMP4 CRPPR 59 CMP5 SKTFNTHPQSTP 60

IX. Peptide-Antisense Oligomer Conjugate Compositions A. Conjugates forSpecific Muscle Treatments

Therapeutic conjugates comprising selected transport peptide sequencesare also provided by the invention. These include conjugates comprisinga carrier peptide (RXRR(B/X)R)₂XB, as described herein, conjugated to anoligonucleotide, e.g., PMO, designed for therapeutic action withinmuscle tissue.

The conjugates may further comprise a targeting moiety effective to bindto tissue specific receptors of a target tissue type, linked to thetherapeutic compound or, preferably, to another terminus of the carrierpeptide. In particularly preferred embodiments, a homing peptide such asdescribed above is conjugated to therapeutic compound or to thecell-penetrating peptide.

For use in treating Duchenne muscular dystrophy, the conjugate compoundcomprises a (RXRR(B/X)R)₂XB, and conjugated to a terminus of thepeptide, an antisense oligonucleotide capable of producing exon skippingin the DMD protein, such as a PMO having SEQ ID NO: 44, to restorepartial activity of the DMD protein.

For use in treating myotonic dystrophy DM1 or DM2, the conjugatecompound comprises an antisense oligonucleotide having 8-30 bases, withat least 8 contiguous bases being complementary to the polyCUG orpolyCCUG repeats in the 3′UTR region of dystrophia myotonica proteinkinase (DMPK) mRNA in DM1 or DM2, respectively, and conjugated to theoligonucleotide, a cell-penetrating peptide having the sequence(RXRR(B/X)R)₂XB, where R is arginine; B is β-alanine; and each X is—C(O)—(CH₂)_(n)—NH—, where n is 4-6. The compound is effective toselectively block the sequestration of muscleblind-like 1 protein(MBNL1) and/or CUGBP in heart and quadricep muscle in a myotonicdystrophy animal model.

For use in treating muscle atrophy, the conjugate compound comprises anantisense oligonucleotide effective to inhibit myostatin expression in asubject, and conjugated to the oligonucleotide, a cell-penetratingpeptide having the sequence (RXRR(B/X)R)₂XB, where R is arginine; B isβ-alanine; and each X is —C(O)—(CH₂)_(n)—NH—, where n is 4-6. Thecompound is effective to inhibit myostatin expression in muscle tissues.

B. Morpholino Oligomers Having Cationic Intersubunit Linkages

In preferred embodiments, as noted above, the antisense oligomer is aphosphorodiamidate morpholino oligonucleotide (PMO). The PMO may includebetween about 20-50% positively charged backbone linkages, as describedbelow and further in PCT Pubn. No. WO 2008036127, which is incorporatedherein by reference.

The cationic PMOs (PMO+) are morpholino oligomers in which at least oneintersubunit linkage between two consecutive morpholino ring structurescontains a pendant cationic group. The pendant group bears a distalnitrogen atom that can bear a positive charge at neutral or near-neutral(e.g. physiological) pH. Examples are shown in FIGS. 1B-C.

The intersubunit linkages in these oligomers are preferablyphosphorus-containing linkages, having the structure:

where

-   W is S or O, and is preferably O,-   X═NR¹R² or OR⁶,-   Y═O or NR⁷,

and each said linkage in the oligomer is selected from:

(a) uncharged linkage (a), where each of R¹, R², R⁶ and R⁷ isindependently selected from hydrogen and lower alkyl;

(b1) cationic linkage (b1), where X═NR¹R² and Y═O, and NR¹R² representsan optionally substituted piperazino group, such thatR¹R²═—CHRCHRN(R³)(R⁴)CHRCHR—, where

each R is independently H or CH₃,

R⁴ is H, CH₃, or an electron pair, and

R³ is selected from H, lower alkyl, e.g. CH₃, C(═NH)NH₂,Z-L-NHC(═NH)NH₂, and {C(O)CHR′NH}_(m)H, where: Z is C(O) or a directbond, L is an optional linker up to 18 atoms in length, preferably up to12 atoms, and more preferably up to 8 atoms in length, having bondsselected from alkyl, alkoxy, and alkylamino, R′ is a side chain of anaturally occurring amino acid or a one- or two-carbon homolog thereof,and m is 1 to 6, preferably 1 to 4;

(b2) cationic linkage (b2), where X═NR¹R² and Y═O, R¹═H or CH₃, andR²=LNR³R⁴R⁵, where L, R³, and R⁴ are as defined above, and R⁵ is H,lower alkyl, or lower (alkoxy)alkyl; and

(b3) cationic linkage (b3), where Y═NR⁷ and X═OR⁶, and R⁷═LNR³R⁴R⁵,where L, R³, R⁴ and R⁵ are as defined above, and R⁶ is H or lower alkyl;

and at least one said linkage is selected from cationic linkages (b1),(b2), and (b3).

Preferably, the oligomer includes at least two consecutive linkages oftype (a) (i.e. uncharged linkages). In further embodiments, at least 5%of the linkages in the oligomer are cationic linkages (i.e. type (b1),(b2), or (b3)); for example, 10% to 80%, 10% to 50%, or 10% to 35% ofthe linkages may be cationic linkages.

In one embodiment, at least one linkage is of type (b1), where,preferably, each R is H, R⁴ is H, CH₃, or an electron pair, and R³ isselected from H, lower alkyl, e.g. CH₃, C(═NH)NH₂, andC(O)-L-NHC(═NH)NH₂. The latter two embodiments of R³ provide a guanidinomoiety, either attached directly to the piperazine ring, or pendant to alinker group L, respectively. For ease of synthesis, the variable Z inR³ is preferably C(O) (carbonyl), as shown.

The linker group L, as noted above, contains bonds in its backboneselected from alkyl (e.g. —CH₂—CH₂—), alkoxy (—C—O—), and alkylamino(e.g. —CH₂—NH—), with the proviso that the terminal atoms in L (e.g.,those adjacent to carbonyl or nitrogen) are carbon atoms. Althoughbranched linkages (e.g. —CH₂—CHCH₃—) are possible, the linker ispreferably unbranched. In one embodiment, the linker is a hydrocarbonlinker. Such a linker may have the structure —(CH₂)_(n)—, where n is1-12, preferably 2-8, and more preferably 2-6.

The use of embodiments of linkage types (b1), (b2) and (b3) above tolink morpholino subunits may be illustrated graphically as follows:

Preferably, all cationic linkages in the oligomer are of the same type;i.e. all of type (b1), all of type (b2), or all of type (b3). Thebase-pairing moieties Pi may be the same or different, and are generallydesigned to provide a sequence which binds to a target nucleic acid.

In further embodiments, the cationic linkages are selected from linkages(b1′) and (b1″) as shown below, where (b1′) is referred to herein as a“Pip” linkage and (b1″) is referred to herein as a “GuX” linkage:

In the structures above, W is S or O, and is preferably O; each of R¹and R² is independently selected from hydrogen and lower alkyl, and ispreferably methyl; and A represents hydrogen or a non-interferingsubstituent on one or more carbon atoms in (b1′) and (b1″). Preferably,the ring carbons in the piperazine ring are unsubstituted; however, theymay include non-interfering substituents, such as methyl or fluorine.Preferably, at most one or two carbon atoms is so substituted.

In further embodiments, at least 10% of the linkages are of type (b1′)or (b1″); for example, 20% to 80%, 20% to 50%, or 20% to 30% of thelinkages may be of type (b1′) or (b1″).

In other embodiments, the oligomer contains no linkages of the type(b1′) above. Alternatively, the oligomer contains no linkages of type(b1) where each R is H, R³ is H or CH₃, and R⁴ is H, CH₃, or an electronpair.

Oligomers having any number of cationic linkages can be used, includingfully cationic-linked oligomers. Preferably, however, the oligomers arepartially charged, having, for example, 5, 10, 20, 30, 40, 50, 60, 70,80 or 90 percent cationic linkages. In selected embodiments, about 10 to80, 20 to 80, 20 to 60, 20 to 50, 20 to 40, or about 20 to 35 percent ofthe linkages are cationic.

In one embodiment, the cationic linkages are interspersed along thebackbone. The partially charged oligomers preferably contain at leasttwo consecutive uncharged linkages; that is, the oligomer preferablydoes not have a strictly alternating pattern along its entire length.

Also considered are oligomers having blocks of cationic linkages andblocks of uncharged linkages; for example, a central block of unchargedlinkages may be flanked by blocks of cationic linkages, or vice versa.In one embodiment, the oligomer has approximately equal-length 5″, 3″and center regions, and the percentage of cationic linkages in thecenter region is greater than about 50%, preferably greater than about70%.

Oligomers for use in antisense applications generally range in lengthfrom about 10 to about 40 subunits, more preferably about 15 to 25subunits. For example, a cationic oligomer having 19-20 subunits, auseful length for an antisense oligomer, may ideally have two to seven,e.g. four to six, or three to five, cationic linkages, and the remainderuncharged linkages. An oligomer having 14-15 subunits may ideally havetwo to five, e.g. 3 or 4, cationic linkages and the remainder unchargedlinkages.

Each morpholino ring structure supports a base pairing moiety, to form asequence of base pairing moieties which is typically designed tohybridize to a selected antisense target in a cell or in a subject beingtreated. The base pairing moiety may be a purine or pyrimidine found innative DNA or RNA (A, G, C, T, or U) or an analog, such as hypoxanthine(the base component of the nucleoside inosine) or 5-methyl cytosine.

As noted above, the substantially uncharged oligonucleotide may bemodified to include one or more charged linkages, e.g. up to about 1 perevery 2-5 uncharged linkages, typically 3-5 per every 10 unchargedlinkages. Optimal improvement in antisense activity is seen where up toabout half of the backbone linkages are cationic. Some, but not maximumenhancement is typically seen with a small number e.g., 10-20% cationiclinkages; where the number of cationic linkages exceeds 50-60%, thesequence specificity of the antisense binding to its target may becompromised or lost.

The enhancement seen with added cationic backbone charges may, in somecase, be further enhanced by distributing the bulk of the charges closeof the “center-region” backbone linkages of the antisenseoligonucleotide, e.g., in a 20-mer oligonucleotide with 8 cationicbackbone linkages, having 70%-100% of these charged linkages localizedin the 10 centermost linkages.

C. Other Oligomer Types

Delivery of alternative antisense chemistries can also benefit from thedisclosed carrier peptide. Specific examples of other antisensecompounds useful in this invention include those in which at least one,or all, of the internucleotide bridging phosphate residues are modifiedphosphates, such as methyl phosphonates, phosphorothioates, orphosphoramidates. Also included are molecules wherein at least one, orall, of the nucleotides contains a 2′ lower alkyl moiety (e.g., C1-C4,linear or branched, saturated or unsaturated alkyl, such as methyl,ethyl, ethenyl, propyl, 1-propenyl, 2-propenyl, or isopropyl).

In other oligonucleotide mimetics, both the sugar and theinternucleoside linkage, i.e., the backbone, of the nucleotide units aremodified. The base units are maintained for hybridization with anappropriate nucleic acid target compound. One such oligomeric compound,an oligonucleotide mimetic that has been shown to have excellenthybridization properties, is referred to as a peptide nucleic acid(PNA). In PNA compounds, the sugar-phosphate backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone.

Modified oligonucleotides may be classified as “chimeric”, e.g.containing at least one region wherein the oligonucleotide is modifiedso as to confer increased resistance to nuclease degradation orincreased cellular uptake, and an additional region for increasedbinding affinity for the target nucleic acid.

EXAMPLES

The following examples are intended to illustrate but not to limit theinvention.

Materials and Methods In Vitro and In Vivo Assays

Nuclear Activity Assay. The effectiveness of each P-PMO conjugate wasdetermined in a splice-correction assay to assess nuclear activity whichutilizes a P-PMO targeted splice site in a plasmid created by aninterruption in the luciferase coding sequence by the human β-globinthalassemic intron 2 which carries a mutated splice site at nucleotide705 (pLuc705). The plasmid is stably transfected in HeLa S3 cells,allowing for easy comparison of the relative efficiency of variouscarrier peptides to deliver PMO (705; 5′-CCT CTT ACC TCA GTT ACA-3′; SEQID NO: 1) capable of restoring splice-correction in cell nuclei. Cellswere cultured in RPMI 1640 medium supplemented with 2 mM L-Glutamine,100 U/mL penicillin, and 10% fetal bovine serum (FBS) at 37° C. in ahumidified atmosphere containing 5% CO₂, and seeded for 20 hours priorto 2 μM P-PMO treatment. All cell treatments with P-PMO were carried outin OptiMEM medium with or without FBS for 24 hours. After celltreatment, restoration of correct splice-correction was measured bypositive readout of luciferase expression in cell lysates on an Flx 800microplate fluorescence-luminescence reader with excitation at 485 nmand emission at 524 nm.

Cell Uptake Assay. The cellular uptake of P-PMO in HeLa pLuc705 cellswas determined using 3′-carboxyfluorescein-tagged P-PMO (P-PMOF) andflow cytometry. Cells were seeded for 20 hours prior to 2 μM P-PMOFtreatment. After treatment, cells were trypsinized to remove any cellmembrane-bound P-PMOF, and washed and resuspended in PBS (Hyclone,Ogden, Utah) containing 1% FBS and 0.2% NaN₃. Cell uptake of P-PMOF wasthen determined by flow cytometry on a FC-500 Beckman Coulter(Fullerton, Calif.) cytometer and data was processed using FCS Express 2software (De Novo Software, Thornhill, Ontario, Canada).

RNA Extraction. Tissue RNA was extracted using Qiagen's RNeasy Mini Kit(Qiagen USA, Valencia, Calif.) per manufacturer's protocol. All isolatedRNA was stored at −80° C.

RT-PCR. Restoration of splice-correction was determined by RT-PCRamplification of EGFP mRNA extracted from tissues harvested from P-PMOtreated EGFP-654 transgenic mice using the Invitrogen SuperScript™ IIIOne-Step RT-PCR System.

Toxicity Assays

The cellular toxicity of P-PMOs was determined bymethylthiazoletetrazolium-survival (MTT), propidium iodine (PI)exclusion, and hemolysis assays, which measured the effects of theP-PMOs on cellular metabolic activity, membrane integrity, and red bloodcell compatibility, respectively.

MTT Analysis. For MTT analysis, cells were seeded at a concentration of9000 cells/well in 96 well plates for 20 hours then treated with P-PMOranging in concentration from 2-60 μM. MTT solution was then added tothe cells for 4 hours and cellular metabolic activity was measured byreading the absorbance of the treatment medium and normalizing theabsorbance of the P-PMO treated samples to the absorbance mean ofuntreated samples. Microscopic images of P-PMO treated cells werevisualized on a Nikon Diaphot inverted microscope (Melville, N.Y.) andprocessed by Magnafire software (Optronics, Goleta, Calif.) forcorrelation with MTT results. All assays were done using HeLa pLuc705cells. Microscopic images of P-PMO treated cells were visualized on aNikon Diaphot inverted microscope (Melville, N.Y.) and processed byMagnafire software (Optronics, Goleta, Calif.) for correlation with MTTresults. All assays were done using HeLa pLuc705 cells.

Propidium Iodine-Exclusion. For PI-exclusion analysis, cells were seededat a concentration of 100,000 cells/well in 12-well plates for 20 hoursthen treated with P-PMO ranging in concentration from 2-60 μM. Cellswere then trypsinized, washed in PBS, and resuspended in PBS containingPI for 15 minutes. Detection of unhealthy or damaged cellular membraneswas done by analyzing cells for PI uptake by flow cytometry.

Red Blood Cell Compatibility. Hemolytic activities in red blood cellsexposed to P-PMO ranging in concentration from 2-60 μM was determinedusing fresh rat blood according to an established method (Fischer, Li etal. 2003).

MDX Mouse Experiments. Experiments using the MDX mouse strain wereperformed essentially as described by Jearawiriyapaisarn, Moulton etal., 2008.

Example 1 Evaluation of Cell Penetrating Peptide Conjugated PMOs in theEGFP-654 Transgenic Mouse Model

A PMO (designated 654; 5′-GCT ATT ACC TTA ACC CAG-3′; SEQ ID NO: 2)designed to restore correct splicing in the enhanced green fluorescentprotein (EGFP) gene was conjugated to various cell penetrating peptides(SEQ ID NOS: 2, 3, 6, 11, 13-14, 19, 20-27) to produce P-PMOs(peptide-conjugated PMOs), which were evaluated in vivo for theirsplice-correction activity and toxicity in the EGFP-654 transgenic mousemodel (Sazani, Gemignani et al. 2002). In this model, the EGFP-654 geneencoding for functional EGFP is interrupted by an aberrantly-splicedmutated intron, and cellular uptake of EGFP-654 targeted P-PMOs can beevaluated by RT-PCR detection of the restored EGFP-654 splice product intissues.

Female EGFP-654 transgenic mice were injected IP once daily for 4consecutive days with saline or a 12.5 mg/kg dose of P-PMO. Posttreatment on day 4, the heart, muscles, liver, kidney, lungs, smallintestine, colon, stomach, mammary gland, thymus, spleen, ovary, skin,bone marrow, and brain were harvested, and extracted RNA was evaluatedby RT-PCR and densitometry of PCR products to determine % correction.Toxicity of P-PMOs was evaluated by measurement of mouse weights overthe course of treatments and immediately prior to necropsy.

Restoration of functional EGFP splice products post treatment withvarious P-PMOs based on RT-PCR analysis of tissues is shown in FIGS.7A-P. Analysis of toxicity based on weights from P-PMO treated miceindicated minimal toxicity (not shown). Optimal carrier peptide uptakefor each tissue (indicated by a *) based on these results is summarizedin Table 2 (see above).

Example 2 Evaluation of PMOs Conjugated to a Cell Penetrating Peptide(CPP) and/or a Muscle Specific Homing Peptide (HP) in the MDX MurineModel of Duschenes Muscular Dystrophy

MDX mice were treated with a series of P-PMO (peptide-conjugated PMOs)containing various combinations of muscle-specific CPPs and HPsconjugated to the M23d antisense PMO. The muscle specific CPP used wasthe “B peptide”, also designated CP06062 (SEQ ID NO: 19), and the musclespecific homing peptide, designated SMP1, was SEQ ID NO: 51. Fourcombinations were tested including CP06062-PMO, MSP-PMO, CP06062-MSP-PMOand MSP-CP06062-PMO, whose compositions are shown in the appendedSequence Table. The M23d antisense PMO (SEQ ID NO: 77) has a sequencetargeted to induce an exon 23 skip in the murine dystrophin gene andrestores functional dystrophin.

The mice received six weekly intravenous injections of a 3 mg/kg dose.The treated mice were sacrificed and various muscle tissues were removedand stained for full-length dystrophin using a dystrophin-specificfluorescent antibody stain.

The results for the CP06062-PMO, MSP-CP06062-PMO and CP06062-MSP-PMOconjugates in five different muscle tissues are shown in FIG. 8. As canbe seen, the dystrophin-specific stain is in much greater evidence forthe MSP-CP06062-PMO compound than for the other two conjugates, with theexception of heart muscle, where the CP06062-MSP-PMO conjugate appearedto have the greatest activity. The observation that the CP06062-MSP-PMOcompound was more effective than the CP06062-PMO conjugate was confirmedby immunoblot and PCR assays (data not shown). In separate experiments(data not shown), an MSP-PMO conjugate induced full-length dystrophin ata level less than the CP06062-PMO conjugate.

Additional examples of muscle-specific delivery of the CP06062-M23dconjugate to tissues of the MDX mouse can be found inJearawiriyapaisarn, Moulton et al., 2008, cited above, which isincorporated herein by reference.

In summary, the combination of the muscle specific homing peptide andmuscle specific cell penetrating peptide significantly improved thedelivery of the M23d antisense peptide as measured in this in vivosystem. The MSP-CP06062-PMO ordering of the peptide moieties wasobserved to induce the highest level of full-length dystrophin and is apreferred embodiment.

Example 3 Improved Cardiac Function in Dystrophin-Deficient Mice by a(RXRRBR)₂XB-Conjugated PMO

It has been demonstrated that a PMO (M23d; SEQ ID NO: 37) targeting thejunction of exon 23 and intron 23 of mouse dystrophin (referred to asM23d hereafter), was able to induce up to functional levels ofdystrophin expression in some skeletal muscles by regular i.v.injections in mdx mice (Alter, J., F. Lou et al. (2006)). However,dystrophin expression induced by PMO required high doses and was highlyvariable between muscles and myofibers in terms of observed efficacy. Ofgreater concern, cardiac muscle seemed to be refractory to the antisensetherapy, failing to produce detectable dystrophin even after repeatedtreatment (seven times at ≈60 mg/kg PMO per injection; Alter, J., F. Louet al. (2006)). Both potency and cardiac delivery represent majorlimitations to antisense therapy as an effective treatment formuscle-specific diseases such like DMD, DM1 and DM2. Because DMDpatients live longer owing to improved multidisciplinary patient care,rescuing dystrophin expression in cardiac muscle becomes more criticalfor their longevity and quality of life. More importantly, restorationof dystrophin only in skeletal muscles may exacerbate the failure ofheart function if dystrophin expression cannot be effectively restoredin cardiac muscle. It is not understood why PMO does not inducedystrophin expression effectively in cardiac muscle even at high dosesbut low delivery efficiency seems to be the most important contributingfactor (Alter, J., F. Lou et al. (2006)). This example describesexperiments using a cell-penetrating peptide-conjugated PMO (SEQ ID NO:62, M23d-CP06062 (SEQ ID NO:37, SEQ ID NO:19); in the MDX mouse model.The results demonstrate the restoration of almost normal levels ofdystrophin in cardiac and other types of muscles bodywide in dystrophicmdx mice, with improvement in muscle strength and cardiac function. Thelatter prevents heart failure under increased workload conditionsinduced by dobutamine. Repeated treatment maintains levels of dystrophinand ameliorates pathology, with significant reduction in levels of serumcreatine kinase without immune response.

To improve the efficiency of exon skipping in muscles, particularly incardiac muscle, several arginine-rich cell-penetrating peptidesconjugated to the same M23d PMO (SEQ ID NO: 62) were tested in the MDXmouse model. The M23d-PMO conjugated to the (RXRRBR)₂XB (SEQ ID NO: 19)(also referred to herein as CP06062 peptide) showed the highestefficiency for skipping exon 23 by i.m. injection in the adult (age 4-5weeks) MDX mouse (FIG. 9).

Strong dystrophin expression was induced in 85% of the fibers in theentire tibialis anterior (TA) muscle after injection of 2 micrograms ofM23d-CP06062 PPMO (FIG. 9). The same amount of unconjugated M23d PMOproduced only 14% dystrophin-positive fibers. A sequence-scrambled PPMO(with the antisense oligomer sequence not complementary to thedystrophin gene but the same base composition as M23d) showed no effecton dystrophin production (not shown). Specific skipping of exon 23 wasconfirmed by RT-PCR and subsequent sequencing. No increases in muscledamage, inflammatory cellular infiltrates, or necrotic fibers wereobserved microscopically in the muscles injected with any of the PPMOsand PMO.

Systemic treatment was investigated by administration of a single doseof 30 mg/kg of M23d-CP06062 PPMO i.v. into adult MDX mice.Administration of this amount of unmodified M23d PMO induced dystrophinexpression in 5% or less of muscle fibers of all skeletal muscles and nodetectable dystrophin in cardiac muscle when examined 2 weeks afterinjection (Alter, J., F. Lou et al. (2006)). In striking contrast,treatment with M23d-CP06062 PPMO produced strong dystrophin expressionin 100% of fibers of all skeletal muscles examined, including the TA,quadriceps, gastrocnemius, abdominal, intercostals, diaphragm, andbiceps (FIGS. 10, B-D). Expression of dystrophin was highly homogeneousthroughout the entire length of the muscles (from tendon to tendon). Infact, the levels of dystrophin expression in the muscles of theM23d-CP06062 PPMO-treated mice were difficult to distinguish from thatin the muscles of normal C57BL mice by immunohistochemical analysis(FIGS. 10, B-D). However, variation in fiber size and specifically thepresence of central nucleation in most muscle fibers were theunmistakable remaining pathology of the mdx mouse. Consistently,near-normal levels (91-100%) of dystrophin were detected by Western blot(FIG. 10, E). The size of the M23d-CP06062 PPMO-induced dystrophin wasindistinguishable from that of the normal dystrophin. Similarly,dystrophin mRNA with exon 23 skipped accounted for 80-86% of RT-PCRproducts in all skeletal muscles (FIG. 10, G). No off-target skipping ofthe neighboring exons was observed. Precise skipping of exon 23 wasconfirmed by sequencing (FIG. 10, H). Restoration of dystrophinexpression also restored the a dystroglycan, a sarcoglycan, and βsarcoglycan on fiber membrane (not shown). Dystrophin expression was notobserved in the muscles of the mdx mice treated with scrambled PPMO(FIGS. 10, B-G).

Importantly, immunohistochemistry demonstrated membrane-localizeddystrophin in 94% of cardiac muscle fibers of mdx mice treated with thesingle dose of M23d-CP06062 PPMO, although the levels of dystrophinvaried (FIG. 10, D). Dystrophin was expressed at near-normal levels inmost areas of the cardiac muscle. A 58% normal dystrophin level wasdemonstrated by Western blot (FIG. 10, E). Consistently, dystrophin mRNAwith exon 23 skipped accounted for 63% of the dystrophin transcript byRT-PCR (FIG. 10, G).

Regular injections of the arginine-rich peptide to maintain or furtherenhance dystrophin expression was investigated. A group of five adultmdx mice received a 3-month treatment with repeated (six times) i.v.injections of 30 mg/kg of M23d-CP06062 PPMO at biweekly intervals. Twoweeks after the last injection, dystrophin expression remained in 100%of muscle fibers in all skeletal muscles, including the diaphragm andsmooth muscles in the small intestine (FIGS. 11, A-C, FIG. 12). Thelevels of dystrophin expression detected by both immunohistochemistryand Western blot in the M23d-CP06062 PPMO-treated mdx mice were againindistinguishable from those in normal C57BL mouse (FIG. 11, D). Thedystrophin mRNA with exon 23 skipped accounted for nearly 90% (85-92%)of total dystrophin mRNA by RT-PCR in all skeletal muscles (FIG. 11, F).

Example 4 Single Low-Dose (RXRRXR)₂XB PPMO Conjugates Restore DystrophinExpression in Muscle and Cardiac Tissue

Single intravenous injections of PPMO conjugates to restore dystrophinexpression systemically was investigated. A 25 mg/kg single injectionadministration protocol was tested with the (RXRRXR)₂XB-M23d PPMOconjugate (SEQ ID NO: 77 conjugated to SEQ ID NO: 11) administered viathe mouse tail vein. Three weeks following single injections, allskeletal muscle fibres immunostained positive for sarcolemmaldystrophin. The intensity of dystrophin expression was near normal inmost skeletal muscle groups analysed, although slightly lower in bicepsas shown (FIG. 13A). Widespread, uniform expression of dystrophinprotein over multiple tissue sections within each muscle group wasdetected in hind limb, fore limb, abdominal wall and diaphragm muscles.Surprisingly, no obvious area-to-area variation was found withinindividual muscle groups as previously reported with the systemicdelivery of naked PMO AOs (Alter, J., F. Lou et al. (2006)). RT-PCRresults revealed almost total exon skipping of the mutated transcriptwith highly effective skipping of mdx dystrophin exon 23 (FIG. 13B) inall skeletal muscles analysed including the diaphragm. Less efficientmolecular correction was observed in heart, where 50% of the mutatedtranscript was found to be exon skipped by RT-PCR. A shorter band wasalso detected in the RT-PCR assay in many analysed tissues, which waslikely to correspond to a skipped transcript lacking exons 22 and 23.Subsequent sequencing of this PCR fragment confirmed that the minortranscript product contained exon 22 and 23 deletions.

To quantify the levels of dystrophin protein restored, western blotanalysis was undertaken, using total protein extracted from all musclegroups including heart, and from normal C57 TA and heart muscle tissuesas positive controls. This indicated that between 25 and 100% of normaldystrophin protein levels had been restored in body-wide skeletalmuscles following the single systemic AO injection. Of particularsignificance were the levels approaching 100% restoration of dystrophinprotein that were detected in distal muscle groups, i.e. TA and biceps,while even in the diaphragm almost 25% of normal dystrophin protein wasrestored (FIG. 13C).

Although the invention has been described with respect to certainembodiments and examples, it will be appreciated that various changes,modifications, and additions may be made without departing from theclaimed invention.

Sequence Table SEQ ID Designation(s) Sequence NO.^(a)Antisense Oligomers 705 5′-CCT CTT ACC TCA GTT ACA-3′ 1 6545′-GCT ATT ACC TTA ACC CAG-3′ 2 Cell-Penetrating Peptides (CPP) R₈RRRRRRRR-XB 3 r₈ rrrrrrrr-XB 4 R₉ RRRRRRRRR-XB 5 (RX)₈RXRXRXRXRXRXRXRX-B 6 (rX)₈ rXrXrXrXrXrXrXrX-B 7 (RX)₇ RXRXRXRXRXRXRX-B 8(RX)₅ RXRXRXRXRX-B 9 (RX)₃ RXRXRX-B 10 (RXR)₄ RXRRXRRXRRXRX-B 11 (rXR)₄rXRrXRrXRrXR-B 12 (rXr)₄ rXrrXrrXrrXr-XB 13 (RB)₈ RBRBRBRBRBRBRBRB-B 14(rB)₈ rBrBrBrBrBrBrBrB-B 15 (RB)₇ RBRBRBRBRBRBRB-B 16 (RB)₅ RBRBRBRBRB-B17 (RB)₃ RBRBRB-B 18 B(3b); CP06062; RXRRBRRXRRBR-XB 19 (RXRRBR)₂XBD(2); RBRBRBRBRBRXRBRX-B 20 (RB)₅RXRBRX-B E(3c); RBRBRBRXRBRBRBRX-X 21(RBRBRBRX)₂X F(3a); X-RBRBRBRXRBRBRBRX 22 X- RB)₃RX(RB)₃RXG(4b); (RBRX)₄B RBRXRBRXRBRXRBRX-B 23 H(4a); RBRBRBRBRXRXRXRX-B 24(RB)₄(RX)₄B I(3d); RXRBRBRXRBRBRBRX-X 25 RX(RB)₂RX(RB)₃ RX-X C(4c);RXRRXRRXRRBR-XB 26 (RXR)₃RBR-XB (RB)₇RX-B RBRBRBRBRBRBRBRX-B 27Oligonucleotide sequences H53A(+39+69) CATTCAACTGTTGCCTCCGGTTCTGAAGGTG28 H53A(+39+62) CTGTTGCCTCCGGTTCTGAAGGTG 29 H53A(+45+69)CATTCAACTGTTGCCTCCGGTTCTG 30 H44A(+85+104) TTTGTGTCTTTCTGAGAAAC 31H44A(−06+14) ATCTGTCAAATCGCCTGCAG 32 H44D(+10−10) AAAGACTTACCTTAAGATAC33 AVI-4657 CTT ACA GGC TCC AAT AGT GGT 34 (hu-exon51) CAG THu.DMD.Exon51. ATT TCT AGT TTG GAG ATG GCA 35 010 GTT TC Hu.DMD.Exon51.GAG CAG GTA CCT CCA ACA TCA 36 012 AGG AA M23d PMOGGCCAAACCTCGGCTTACCTGAAAT 37 AVI-4658 CTCCAACATCAAGGAAGATGGCATTT 38(hu-exon 51) CTAG Human Myostatin ACTCTGTAGGCATGGTAATG 39 SD1Human Myostatin CAGCCCATCTTCTCCTGG 40 SD2 Human MyostatinCACTTGCATTAGAAAATCAG 41 SA2 Human Myostatin CTTGACCTCTAAAAACGGATT 42 SA3Human Myso- GAGTTGCAGTTTTTGCATG 43 statin-AUG CAG25AGCAGCAGCAGCAGCAGCAGCAGCA 44 CAG22 AGCAGCAGCAGCAGCAGCAGCA 45 CAG19AGCAGCAGCAGCAGCAGCA 46 CAG12 AGCAGCAGCAGC 47 CCAG24AGCCAGCCAGCCAGCCAGCCAGCC 48 CUG39 CUGCUGCUGCUGCUGCUGCUGCUG 49CUGCUGCUGCUGCUG CCUG40 CCUGCCUGCCUGCCUGCCUGCCUG 50 CCUGCCUGCCUGCCUG SEQID Homing peptides Peptide Sequence (NH₂ to COOH) NO. Skeletal Muscle-ASSLNIA 51 SMP1 SMP2 SLGSFP 52 SMP3 SGASAV 53 SMP4 GRSGAR 54 SMP5TARGEHKEEELI 55 Cardiac Muscle- WLSEAGPVVTVRALRGTGSW 56 CMP1 CMP2VTVRALRGTSW 57 CMP3 VVTVRALRGTGSW 58 CMP4 CRPPR 59 CMP5 SKTFNTHPQSTP 60Conjugate compounds (RXRRBR)₂XB- RXRRBRRXRRBR-XB- 61 4658GGCCAAACCTCGGCTTACCTGAAAT (RXRRBR)₂XB- RXRRBRRXRRBR-XB- 62 M23d PMOGGCCAAACCTCGGCTTACCTGAAAT (RXRR(B/X)R)₂X RXRRBRRXRRBR-B- 63 B-myoGGCCAAACCTCGGCTTACCTGAAAT (RXRR(B/X)R)₂X RXRRBRRXRRBR-XB- 64 BmyoGGCCAAACCTCGGCTTACCTGAAAT (RXRR(B/X)R)₂X RXRRBRRXRRBR-XB- 65 B CAG25AGCAGCAGCAGCAGCAGCAGCAGCA (RXRR(B/X)R)₂X RXRRBRRXRRBR-XB- 66 B CCAG24AGCCAGCCAGCCAGCCAGCCAGCC ^(a)In SEQ ID Nos. 3-27, sequences assigned toSEQ ID NO. do not include the linkage portion (X, B, or XB).

It is claimed:
 1. An antisense compound for use in treating myotonicdystrophy DM1 or DM2, comprising an antisense oligonucleotide having8-30 bases, with at least 8 contiguous bases being complementary to thepolyCUG or polyCCUG repeats in the 3′UTR region of dystrophia myotonicaprotein kinase (DMPK) mRNA in DM1 or DM2, respectively, and conjugatedto the oligonucleotide, a cell-penetrating peptide having the sequence(RXRR(B/X)R)₂XB, where R is arginine; B is β-alanine; and each X is—C(O)—(CH₂)_(n)—NH—, where n is 4-6. where the compound is effective toselectively block the sequestration of at least one of muscleblind-like1 protein (MBNL1) and CUGBP in heart and quadricep muscle in a myotonicdystrophy animal model.
 2. The antisense compound of claim 1, whereinthe cell penetrating peptide has the form (RXRRBR)₂XB and X is—C(O)—(CH₂)₆—NH—.
 3. The antisense compound of claim 2, wherein theoligonucleotide is a phosphorodiamidate oligonucleotide (PMO) havingbetween 12-30 bases, and at least 12 contiguous bases that arecomplementary to the polyCUG repeats in the 3′UTR region of dystrophiamyotonica protein kinase (DMPK) mRNA in DM1.
 4. The antisense compoundof claim 2, wherein the oligonucleotide is a phosphorodiamidateoligonucleotide (PMO) having between 12-30 bases, and at least 12contiguous bases that are complementary to the polyCCUG repeats in the3′UTR region of dystrophia myotonica protein kinase (DMPK) mRNA in DM2.5. The antisense compound of claim 1, wherein the cell penetratingpeptide has the form (RXRRXR)₂XB and X is —C(O)-(CH₂)₆—NH—.
 6. Theantisense compound of claim 5, wherein the oligonucleotide is aphosphorodiamidate oligonucleotide (PMO) having between 12-30 bases, andat least 12 contiguous bases that are complementary to the polyCUGrepeats in the 3′UTR region of dystrophia myotonica protein kinase(DMPK) mRNA in DM1.
 7. The antisense compound of claim 5, wherein theoligonucleotide is a phosphorodiamidate oligonucleotide (PMO) havingbetween 12-30 bases, and at least 12 contiguous bases that arecomplementary to the polyCCUG repeats in the 3′UTR region of dystrophiamyotonica protein kinase (DMPK) mRNA in DM2.
 8. A method of targeting asystemically administered antisense oligonucleotide to heart muscletissue in a mammalian subject, where the oligonucleotide is directedagainst the polyCUG or polyCCUG repeats in the 3′UTR region ofdystrophia myotonica protein kinase (DMPK) mRNA in DM1 or DM2,respectively, comprising conjugating to the oligonucleotide, acell-penetrating peptide having the sequence (RXRR(B/X)R)₂XB, where R isarginine; B is β-alanine; and each X is independently a neutral linearamino acid —C(O)—(CH₂)₆—NH—, where n is 4-6.
 9. The method of claim 8,wherein the selective targeting of the oligonucleotide to heart andquadracep muscle is evidenced by the reversal of sequestration ofmuscleblind-like 1 protein (MBNL1) in the heart and quadricep muscletissue in a PMO-treated animal model having known MBNL1 sequestration.10. The method of claim 8, wherein the cell penetrating peptide has theform (RXRRBR)₂XB and X is —C(O)—(CH₂)₆—NH—.
 11. The method of claim 10,wherein the oligonucleotide is a phosphorodiamidate oligonucleotide(PMO) having between 12-30 bases, and at least 12 contiguous bases thatare complementary to (i) the polyCUG repeats in the 3′UTR region ofdystrophia myotonica protein kinase (DMPK) mRNA in DM1, or (ii) thepolyCCUG repeats in the 3′UTR region of dystrophia myotonica proteinkinase (DMPK) mRNA in DM2.
 12. The method of claim 8, wherein the cellpenetrating peptide has the form (RXRRXR)₂XB and X is —C(O)—(CH₂)₆—NH—.13. The method of claim 12, wherein the oligonucleotide is aphosphorodiamidate oligonucleotide (PMO) having between 12-30 bases, andat least 12 contiguous bases that are complementary to (i) the polyCUGrepeats in the 3′UTR region of dystrophia myotonica protein kinase(DMPK) mRNA in DM1, or (ii) the polyCCUG repeats in the 3′UTR region ofdystrophia myotonica protein kinase (DMPK) mRNA in DM2.
 14. A method oftreating mytonic dystrophy DM1 or DM2 in a mammalian subject, comprisingadministering to the subject, an antisense compound comprising anantisense oligonucleotide having 8-30 bases, with at least 8 contiguousbases being complementary to the polyCUG or polyCCUG repeats in the3′UTR region of dystrophia myotonica protein kinase (DMPK) mRNA in DM1or DM2, respectively, and conjugated to the oligonucleotide, acell-penetrating peptide having the sequence (RXRR(B/X)R)₂XB, where R isarginine; B is β-alanine; and each X is —C(O)—(CH₂)_(n)—NH—, where n is4-6, and repeating said administering at least once every one week to 3months.
 15. The method of claim 14, for treating DM1 in a mammaliansubject, wherein the cell penetrating peptide has the form (RXRRBR)₂XB,X is —C(O)—(CH₂)₆—NH—, and the oligonucleotide is a phosphorodiamidateoligonucleotide (PMO) having between 12-30 bases, and at least 12contiguous bases that are complementary to the polyCUG repeats in the3′UTR region of dystrophia myotonica protein kinase (DMPK) mRNA in DM1.16. The method of claim 14, for treating DM2 in a mammalian subject,wherein the cell penetrating peptide has the form (RXRRBR)₂XB, X is—C(O)—(CH₂)₆—NH—, and the oligonucleotide is a phosphorodiamidateoligonucleotide (PMO) having between 12-30 bases, and at least 12contiguous bases that are complementary to the polyCCUG repeats in the3′UTR region of dystrophia myotonica protein kinase (DMPK) mRNA in DM2.17. The method of claim 14, for treating DM1 in a mammalian subject,wherein the cell penetrating peptide has the form (RXRRXR)₂XB, X is—C(O)—(CH₂)₆—NH—, and the oligonucleotide is a phosphorodiamidateoligonucleotide (PMO) having between 12-30 bases, and at least 12contiguous bases that are complementary to the polyCUG repeats in the3′UTR region of dystrophia myotonica protein kinase (DMPK) mRNA in DM1.18. The method of claim 14, for treating DM2 in a mammalian subject,wherein the cell penetrating peptide has the form (RXRRXR)₂XB, X is—C(O)—(CH₂)₆—NH—, and the oligonucleotide is a phosphorodiamidateoligonucleotide (PMO) having between 12-30 bases, and at least 12contiguous bases that are complementary to the polyCCUG repeats in the3′UTR region of dystrophia myotonica protein kinase (DMPK) mRNA in DM2.19. The method of claim 14, wherein said administering is by intravenousor subcutaneous injection to the subject, at a dose between 1-5 mg/kgbody weight antisense compound.
 20. The method of claim 14, wherein saidadministering is repeated repeating step is continued at regularintervals of every one to three months, and further includes monitoringthe patient during the treatment period for improvement in skeletal orheart muscle performance.
 21. The method of claim 14, wherein saidadministering is repeated repeating step is continued at regularintervals of every one to three months, and further includes monitoringthe patient during the treatment period for improvement in heartconduction properties.
 22. The method of claim 20, wherein saidadministering is repeated repeating step is continued at regularintervals of every one to three months, and further includes monitoringthe patient during the treatment period for reduction in serum creatinekinase.